To evaluate cytotoxicity, RDV hydrogels were extracted using 5 mL of DMEM/F12 medium to obtain a standard leach liquor [25 (link)]. The HCECs were seeded onto a 96-well plate with 1 × 104 cells per well and then co-cultured with 100 μL leaching liquor. The morphological appearance of HCECs was observed using a DM IL LED microscopy (Leica, Germany) to obtain a sense of immediacy. Cell proliferation capacity was measured following the instructions of the Cell Counting Kit-8 (https://www.dojindo.eu.com/TechnicalManual/Manual_CK04.pdf , accessed on 12 December 2021, CCK-8, Dojindo, Japan).
The rate of apoptosis in HCECs was measured in all groups using the TUNEL assay. This assay identified DNA breaks by labeling the 3′-OH terminals of DNA with modified nucleotides to TdT or terminal nucleotidyltransferase. Briefly, HCECs were cultured on slides and then treated with the leach liquor. After an incubation period of 48 h, the existence of apoptotic cells in the samples was investigated using a DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) in accordance with the protocol of the manufacturer.
The rate of apoptosis in HCECs was measured in all groups using the TUNEL assay. This assay identified DNA breaks by labeling the 3′-OH terminals of DNA with modified nucleotides to TdT or terminal nucleotidyltransferase. Briefly, HCECs were cultured on slides and then treated with the leach liquor. After an incubation period of 48 h, the existence of apoptotic cells in the samples was investigated using a DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) in accordance with the protocol of the manufacturer.
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